Download Genes The Yersinia MLST scheme uses fragments of the following

Genes
The Yersinia MLST scheme uses fragments of the following seven house-keeping genes:
aarF - putative ubiquinone biosynthesis protein UbiB
dfp - bifunctional phosphopantothenoylcysteine decarboxylase/phosphopantothenate synthase
galR - DNA-binding transcriptional regulator
glnS - glutaminyl-tRNA synthetase
hemA - glutamyl-tRNA reductase
rfaE - bifunctional heptose 7-phosphate kinase/heptose 1-phosphate adenyltransferase
speA - arginine decarboxylase
PCR amplification
The thermal cycler was set up as follows: stage 1 - initial denaturation 94 ̊C for 5 minutes, stage 2 denaturation 94 ̊C for 30 seconds, stage 3 – annealing temperature dependent upon primer set for 30seconds,
stage 4 – elongation 72 ̊C for 30 seconds, stage 5 – 30 repeats of stages 2 to 4, stage 6 – final elongation
72 ̊C for 5 minutes, and final hold at 4 ̊C. The PCR reactions were carried out using the GoTaq® Flexi DNA
polymerase kit (Promega, catalogue number:M8301), and dNTPs (Promega, catalogue number: U1330), in
the quantities and concentrations as follows for one reaction: 5 μl 1.5 mM MgCl2, 5 μl PCR reaction buffer,
2 μl 10 μM dNTPs, 0.3 μl 5 U/μM Taq DNA polymerase, 40 μl sterilised distilled water, 0.5 μl 10 pmol of
each forward and reverse primers, 1 μl ~10 ng/μl DNA. The amplification product was then purified using
ExoSAP-IT (Affymetrix, catalogue number: 78201 1ML) and submitted for Sanger sequencing in duplicate
to obtain independent forward and reverse reactions.
MLST primers
The primers were used for both the amplification and sequencing reactions. The second set of primers for
rfaE (labelled rfaE*) were designed for Y. enterocolitica serotype O:3 strains originating from Germany,
due to low quality sequence produced with the original primers.
MLST
gene
aarF
dfp
galR
glnS
hemA
speA
rfaE
rfaE*
PCR
MLST
product
region
length (bp) length (bp)
5'-ttccatgcagatatgcacc-3'
3'-ccactcactaatagtgtagc-5'
650
500
5'-gatccggtacgctttatcag-3'
3'-cataacggctgacaatctcg-5'
547
455
5'-attggtaacggttaccatg-3'
3'-gttgggctgaacatattggt-5'
648
500
5'-gaatcatgtatccgtgatg-3'
3'-gcacagaaataaccttcac-5'
557
442
5'-atgactctgctcgcattagg-3'
3'-cggttggcaataatcatatg-5'
602
490
5'-atgtctgatgataacttgatt-3'
3'-cagataaactttatggccc-5'
550
452
5'-atgaaagtcactctgcctga-3'
3'-atcactgcctttaggatc-5'
509
429
Alternate rfaE primers specific for Y. enterocolitica serotype O:3
5'-atgaaagtcacgctgcctga-3'
3'-ggatagatttggtgtcagtagc-5'
550
429
Forward Primer
Reverse Primer
Annealing
Temperature
(°C)
52
59
59
56.5
54
55.5
55.5
57.5
Reference MLST regions from Y. enterocolitica 8081
>8081_aarF
CACTAAATAAAGCAGATAAGCGCTATCTTGCTGAAAACTTTATTGCTTTCTTTAATCGGGATTA
TCGGCGGGTTGCAGAGCTGCATGTTGACTCTGGCTGGGTTCCTTGTGATACCAATGTTGAAGAT
TTTGAATTCGCTATCCGTACCGTCTGCGAACCAATTTTTGAAAAACCACTAGCCGAAATATCCT
TTGGCCATGTGTTATTGAATCTCTTTAATACCGCACGCCGTTTTAATATGGAAGTACAACCACA
GTTGGTTTTACTGCAAAAAACCTTGTTGTATGTCGAAGGCTTAGGGCGTCAACTTTATCCTCAG
CTTGACCTTTGGACGACTGCGAAGCCTTTCCTGGAAAGCTGGTTACGAGATCAAGTTGGGTTGC
CTGCGGTTATTCGTGCATTGAAAGAAAAAGCACCATTTTGGGCTGAAAAATTTCCTGAGCTGCC
AGAGCTTGTGTATGACAGTTTGCAACAGCATAAGCTATTACAGCAAAGTGTG
>8081_dfp
TGCCAGAGGGGCAAAAGTTACTCTGGTTGCCGGGCCAGTGATGCTCCCTACTCCAGCGGGAGT
GAATCGTATTGATGTTGTCAGTGCGCTAGAAATGCAGCAAACAGTTCAGAACTTAGCAGCCCA
GCAGAATATTTTTATTTCTTGTGCTGCGGTAGCGGATTACCGCGCTGAGCAGGTTTCTGACGAG
AAAATAAAAAAACAGGGCGATGAGATCACCCTTAAGTTGGTGAAAAATCCAGACATTGTCGCT
GGAGTCGCTTCGATGACTAAAAAACGTCCATTTGTTGTTGGATTTGCTGCCGAAACCCAGAATG
TGGAAGAATACGCGCGACAAAAGCTAGCGCGGAAAAATCTGGATCTTATTTGCGCTAATGATG
TATCGCTCGCAGAGCATGGTTTTAACAGTGATACTAATGCCTTGCACCTTTTTTGGCCGACTGG
CGAGAAACGTT
>8081_galR
AAAATTATCTGATGAAGAATTGACGTCATTAATGCAGCAAATCCCTGGCATGGTGTTGATTAAC
CGCACCCTCCCCGGTTTTGAAACCCGTTGTGTGGCGTTGGATGACCGTTATGGTGCCTGGCTGG
GAACCCGCCATCTGATTCAGCATGGGCATAAACGAATAGCGATTATTTGCTCCAATCATCAAAT
TTCTGATGCCACCGATCGTCTGCAAGGTTATCTGGATGCTCTGGAAGAGTTTGGTATTGCGGTT
GATGACCGTTTAATTTCCTACGGTACGCCGGATGAAATCGGCGGTGAACAAGCCATGACTGAC
TTACTTGGCCGAGGCCGAAACTTCACTGCGGTCACTTGTTATAACGACTCAATGGCAGCGGGA
GCATTGTCCGTACTCAGCGATAACAGTATCGAAGTGCCACAAGAGATCTCACTGATCGGTTTTG
ACGATGTGTTGATCTCCCGTTATCTGCGCCCGCGGCTGACCACCATTCGCTATC
>8081_glnS
GAGAATCGTGCGGCAGGGGAAGAGTGGCTGACCATGCCAAATCATCCGAATAATCCGGATATG
GGCACCCGTCAGGTACCTTTCGACAGTGAGATTTATATCGATCGCGCTGATTTTCGTGAAGAAG
CCAATAAGCAATACAAGCGTCTGGTGCTGGGTAAAGAAGTGCGCCTGCGTAATGCTTATGTCA
TCAAAGCCGAGCGTGTTGAGAAAGACGCCGAAGGTCATGTCACGACACTTTATTGCAGCTATG
ACGCAGAAACTTTAAATAAAGACCCTGCCGACGGCCGTAAAGTGAAAGGGGTTATCCATTGGG
TTTCTGTTAAGCATGCATTGCCTGCTGAAATCCGTTTATATGACCGTTTGTTTAGTGTACCAAAC
CCAGCGGCGGCGGAAGACTTCTTATCTACCATCAACCCAGAGTCTTTGATTATCCGTCAGG
>8081_hemA
CACCTGAATCGATCGATCAGGCACTTGCTAGCTTGCTCCAACAGCCGTTGGTGCAGGGCGGTGT
CGTGTTGTCTACGTGTAACCGAACTGAGTTATATCTCAGCGTAGAACAGCAAGAAAACCTGCA
CGAACAACTGATTGCCTGGCTGTGTAACTACCATAAACTCAGCCCGGATGAAGTAAAAAAAAG
CCTCTATTGGCATCATGGCAATGACGCAGTTAGCCATCTGATGCGCGTTGCCAGCGGGCTGGAC
TCGCTGGTGTTGGGCGAGCCACAAATTCTGGGACAGGTGAAAAAAGCCTTTGCAGAGTCGCAA
CGCGAGCAATCGCTCTCTGGCGAATTGGAGCGGTTATTCCAAAAAACATTTTCGGTCGCCAAA
CGGGTTCGCACAGAAACTGAAATTGGTGCTAGTGCTGTTTCGGTGGCTTTCGCTGCCTGTACCT
TGGCACGGCAAATCTTTGAGTCACTGTCAGAGCTGAATGTATTGTT
>8081_rfaE
TGTTAGACCGTTATTGGTATGGCCCAACCAGCCGCATTTCACCAGAAGCACCGGTGCCGGTTGT
CAAAGTCGATACCATCGAAGAACGCCCCGGTGGTGCCGCGAACGTCGCGATGAACATCGCCTC
CCTTGGCGCAATCTCCCGGTTAGTCGGGTTGACGGGTATTGATGATGCCGCACGCGCACTGACC
AGCAAGCTAAACGAAGTGCAGGTGCGTTGTGATTTTGTCTCGGTGCCGACTCACCCAACCATC
ACCAAGTTGCGGGTACTTTCTCGTAACCAGCAGCTCATCCGTCTGGATTTCGAAGAAGGTTTTG
ATGGTGTTGACCCTCAACCTATCTTTGAGCGCATTCAACAAGCGCTGCCACAGATTGGTGCTCT
GGTGCTGTCCGACTATGCCAAAGGTGCATTGAACAGTGTGCAGACGA
>8081_speA
TGAATGATCGTAATGCCAGCAAGATGCTGAGCACGTATAACGTCGCCTACTGGGGGGGTAACT
ATTATGACGTCAATGAACTCGGTCATATCAGTGTTTGTCCTGATCCGGATGTTCGTGAGGCACG
TGTCGATCTAGCTGAATTGGTCAAAAAGATGCAGCTTGAGCAGGGGCAACGCCTGCCTGCGTT
ATTCTGTTTCCCGCAAATTTTACAGCATCGCCTGCGCTCAATTAATGGCGCATTTAAACGTGCA
CGTGAGTCATTTGGCTATGAAGGCGATTACTTCCTGGTTTACCCAATTAAAGTCAATCAGCATC
GCCGGGTTATTGAGTCACTGGTTAATTCCGGTGAGCCATTAGGGCTGGAAGCCGGTTCTAAAG
CCGAAATGATGGCGGTTTTGGCACATGCCGGTATGACTCGCTCGGTCATCGTTTGTAATGGCTA
TAAAGAC